Cells (1×105/ml) were fixed in 4% paraformaldehyde for 20 min at room temperature and permeabilized using 0.1% Triton X-100 for 20 min. Samples were blocked in goat serum (Wuhan Boster Biological Technology, Ltd.; cat. no. AR009) for 30 min and subsequently incubated with the following primary antibodies: Anti-β-catenin (Wanleibio Co., Ltd.; cat. no. WL0962a; 1:200) and anti-mono-ADP-ribose binding reagent (EMD Millipore; cat. no. MABE1076; 1:200) at 4°C overnight. The following day, cells were washed in PBS and incubated with Cy3-conjugated secondary antibody (ProteinTech Group, Inc.; cat. no. SA00009-2; 1:200) in the dark for 1 h at room temperature. DAPI (Wuhan Boster Biological Technology, Ltd.) was used for nuclear staining (5 min at room temperature). The images were captured using ZOETM Fluorescent Cell Imager (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ software (version 1.48; National Institutes of Health) The experiment was repeated three times.
Goat serum
Manufactured by Wuhan Boster Biological Technology
Goat serum is a biological product derived from the blood of healthy goats. It contains a complex mixture of proteins, hormones, and other biomolecules found in the goat's blood. Goat serum is commonly used as a supplement in cell culture media to support the growth and maintenance of various cell types in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Leica stellaris 5, by Leica Microsystems (2 mentions)
Anti rrs1, by Abcam (2 mentions)
Anti mono adp ribose binding reagent, by Merck Group (1 mentions)
Cy3 conjugated secondary antibody, by Proteintech (1 mentions)
Zoetm fluorescent cell imager, by Bio-Rad (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Boster Biological Technology (1 mentions)
Pm20 automatic microscope, by Olympus (1 mentions)
Anti lamin b1, by Proteintech (1 mentions)
Anti c myc, by Thermo Fisher Scientific (1 mentions)
Anti rpl11, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Paraformaldehyde (pfa), by Wuhan Boster Biological Technology (1 mentions)
Ti sr, by Nikon (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Bcl2 associated x (bax), by Wuhan Sanying Biotechnology (1 mentions)
Caspase 3, by Abcam (1 mentions)
Bcl 2, by BD (1 mentions)
Ab205, by Abcam (1 mentions)
24 well plate, by Corning (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
9 protocols using goat serum
1
Immunofluorescent Analysis of β-Catenin and ADP-Ribose
2
Immunohistochemical Analysis of TNIK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC was conducted according to the manufacturer's protocol. Briefly, the sections were deparaffinized, processed for antigen recovery, blocked in goat serum (Wuhan Boster Biological Technology, Wuhan, China) and incubated in TNIK antibody (sc-377215, mouse polyclonal antibody, 1:50) overnight at 4 °C. The next day, the samples were incubated in goat anti-mouse secondary antibody for 30 min at 37 °C. Counterstaining was carried out with Harris's hematoxylin. For negative controls, the primary antibodies were replaced with PBS. Ten visual field images were randomly obtained from every section using an OLYMPUS PM20 automatic microscope (Olympus, Japan) and TCFY-2050 (Yuancheng Inc., China) pathology system.
3
Immunofluorescence Analysis of Nucleolar Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BT549 cells were seeded onto a round coverslip (Biosharp; cat. no. BS-14-RC) at the density of 2,000 cells/well. After 24 h, the cells were fixed with 4% paraformaldehyde for 10 min, permeabilized for with 2% Triton X-100 10 min, blocked in 10% goat serum (Wuhan Boster Biological Technology, Ltd.; cat. no. AR0009) for 30 min (the above steps were carried out at room temperature) and then incubated overnight with anti-RRS1 (1:200, Abcam; cat. no. ab188161), anti-RPL11 (1:200, Proteintech; cat. no. 16277-1-AP), anti-c-Myc (1:50, Thermo, MA5-12080), anti-NPM (1:200, Proteintech; cat. no. 60096-1-Ig), anti-Lamin B1 (1:200, Proteintech 12987-1-AP) and anti-Fibrillarin/U3 RNP (1:200, ABclona; cat. no. A0850) at 4°C. Subsequently, the cells were incubated with the appropriate fluorescent secondary antibody (1:200, ABclona; cat. no. AS039 and AS011) for 1 h at room temperature. Nuclei were stained using DAPI (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. C0065) for 10 min at room temperature according to the manufacturer's protocol. The stained cells were viewed with a laser-scanning confocal microscope (Leica Stellaris 5; Leica Microsystems GmbH), 3 fields of view were randomly selected, observed and images captured at ×200 and ×630 magnification.
4
Immunofluorescence Staining of YAP1 and JAG1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 7×104 cells were seeded into 24-well plates with a coverslip on the bottom and incubated at 37°C in 5% CO2. After 8 h, the cells on the coverslips were fixed with 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd.) for 10 min at room temperature and permeabilized for 20 min in 0.1% Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd.). After blocking in goat serum (Wuhan Boster Biological Technology, Ltd.) for 30 min, slides were incubated with a primary antibody overnight at 4°C. Subsequently, slides were incubated with a goat anti-rabbit immunoglobulin G (IgG) FITC-conjugated secondary antibody (1:50 dilution; cat. no. SA00003-2; ProteinTech Group, Inc.) for 1 h at room temperature. Slides were subsequently counterstained with DAPI (cat. no. C1002; Beyotime Institute of Biotechnology) in the dark for 5 min at room temperature. The following primary antibodies were used for immunofluorescence staining: YAP1 (1:200 dilution; cat. no. GTX129151; GeneTex, Inc.) and JAG1 (1:100 dilution; cat. no. GTX48691; GeneTex, Inc.). Immunofluorescence images were acquired using an inverted fluorescence microscope (Ti-SR; Nikon Corporation; magnification, ×100).
5
Immunohistochemical Analysis of Caspase-3 in Murine Lung
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Left lung tissues of mice were fixed in 4% formalin buffer and embedded in paraffin. The paraffin blocks were then serially sectioned into 4-μm-thick slices and subjected to IHC staining. The sections were deparaffinized in xylene for 25 min and rehydrated in ethanol for 5 min. The sections were incubated in 3% H2O2 for 10 min; 10 mM sodium citrate buffer was used for antigen retrieval in a microwave oven for 3 min at high power and 15 min at low power. The sections were then blocked in goat serum (Wuhan Boster Biological Technology, Wuhan, China) for 30 min at room temperature. Rabbit antibody against caspase3 (1:500; Proteintech, Wuhan, China) was used for IHC staining overnight at 4 °C, followed by a 30 min incubation with a secondary goat anti-rabbit antibody at 37 °C. The sections were washed in PBS and incubated with 3,3-diami-nobenzidine (DAB, Zhongshan Golden Bridge, Beijing, China) for 3 min. Slides were counterstained with hematoxylin before microscopic analysis.
6
Immunofluorescence Analysis of Apoptosis Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells mounted on slides were placed in a culture plate and washed with PBS three times. The cells were then fixed with 4% paraformaldehyde for 15 min at 4°C and incubated with 0.5% Triton X-100 at room temperature for 20 min. Subsequently, cells were blocked with 5–10% goat serum (Wuhan Boster Biological Technology, Ltd.) at room temperature for 30 min. Cells were subsequently incubated overnight at 4°C with primary antibodies against NISCH (dilution, 1:50; cat. no. 558262; BD Biosciences), Bcl-2 (dilution, 1:50; cat. no. 12789-1-AP; Wuhan Sanying Biotechnology), Bax (dilution, 1:250; cat. no. ab32503; Abcam) and caspase-3 (dilution, 1:50, cat. no. 19677-1-AP; Wuhan Sanying Biotechnology). Immunofluorescence was generated by incubating samples with the Cy3-conjugated secondary antibodies goat anti-rabbit IgG (dilution, 1:100; cat. no. BA1032) or goat anti-mouse IgG (1:100; cat. no. BA1031; both Wuhan Boster Biological Technology, Ltd.) for 1 h at 37°C in the dark. DAPI (5 µg/ml) was added to counterstain the nuclei, and the slides were incubated for 5 min at room temperature in the dark before being washed with PBS to remove excess DAPI. Images were obtained using a fluorescence microscope (Olympus BX53; Olympus Corporation, Tokyo, Japan).
7
Visualization of F-actin in Hippocampal Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glass slides (14×14-mm) were placed into 24-well plates (Corning Life Sciences, Corning, NY, USA). Differentially treated hippocampal neuronal cells were cultured on 14×14-mm glass slides for 48 h using the aforementioned procedure. After 48 h, the cells were fixed on 14×14-mm glass slides with 4% paraformaldehyde for 30 min at room temperature then washed with PBS. Following blocking with 3% goat serum (Wuhan Boster Biological Technology, Ltd.) for 30 min at room temperature. The cells were incubated with antibodies specific for F-actin (1:100; ab205; Abcam, Cambridge, UK) overnight at 4°C. Following three washes with PBS, the cells were incubated with goat anti-mouse IgG antibodies (Alexa Fluor488; cat. no. 4408S, Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at 37°C and then the slides were mounted by adding DAPI-Fluoromount-G (1:1,000; Wuhan Sanying Biotechnology, Inc., Wuhan, China) for 5 min at room temperature. Subsequently, cells were examined under a confocal laser scanning microscope (magnification, ×630). ImageJ software 1.45s (National Institutes of Health) was used to analyze the results.
8
Pluripotency and Cell Adhesion Analysis of Blastocysts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On day 6, a number of the blastocysts from the single blastomeres were stained with 2 µg/ml Hoechst-33342 (Sigma-Aldrich) to count the numbers of cells. On day 7, immunofluorescence analysis was performed to detect the expression levels of the pluripotency marker octamer-binding transcription factor (Oct)3/4 and E-cadherin in the blastocysts cultured from single blastomeres. Blastocysts were fixed with 4% formaldehyde at room temperature (RT) and permeabilized with 0.2% Triton-X100 (Sigma-Aldrich) in PBS for 20 min, prior to blocking with 10% goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China) in PBS for 1 h. Subsequently, the blastocysts were incubated with the following primary antibodies overnight at 4°C: Polyclonal rabbit anti-human OCT3/4 (1:200; 18976; Abcam, Cambridge, UK) and polyclonal rabbit anti-human E-cadherin antibody (1:100; 15148; Abcam). The blastocysts were then incubated with a fluorescent-conjugated secondary antibody (goat anti-rabbit IgG fluorescein isothiocyanate; 1:40; 6717; Abcam) for 1 h at RT. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich). Specimens were observed using Zeiss LSM 510 Meta Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany).
9
Immunofluorescent Visualization of GRP78 and RRS1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 and BT-549 cells were cultured at a density of 2000 cells/well on circular coverslips (Biosharp; cat. no. BS 14 RC). After treatment with a 4% PFA solution for 20 min, the mounted coverslips were washed three times with PBS. Following fixation, they were washed with phosphate-buffered saline solution (PBST) three times. A blocking treatment with 10% goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China; cat. no. AR0009) was performed after cell infiltration with a 0.5% Triton X-100 solution for 10 min. Subsequently, coverslips were incubated overnight at the temperature of 4°C with anti-GRP78 (1:200, Proteintech; cat. no. 66574-1-Ig) and anti-RRS1 (1:200, Abcam; Catalog number ab188161) antibodies. The next day, cells were further treated with the appropriate fluorescent secondary antibodies (1:200, ABclonal; cat. no. AS039 and AS011) at room temperature for one hour, followed by three washes with PBST. Subsequently, 4',6-diamidino-2-phenylindole (DAPI) staining (Epizyme, Shanghai, China) was used to stain the nucleus, accompanied with an additional 10 min of incubation at ambient temperature, avoiding light. Finally, the cells were visualized and documented utilizing a laser confocal microscopy (Leica Stellaris 5; Leica Microsystems GmbH, Wetzlar, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!